The goal of this R03 application is to develop a novel method to measure DSB repair at endogenous loci, using a next-generation sequencing platform and automated alignment program that was recently developed in our laboratory. This method is unique from previously described assays, because it does not require the integration of artificial sequences into the genome, and it does not rely on a fluorescent protein as a reporter. As such, our assay could be rapidly applied to many different cell lines, and even primary tumor cultures in the future. Thus, our approach will have uses for both basic science and clinical applications. DSBs are a serious threat to genomic integrity and cell survival, and thus complex pathways have evolved which can rapidly detect DNA breaks, activate cell cycle checkpoints to arrest growth, and then repair these lesions. In addition, tumor cell survival often is dependent on the repair of DSBs induced by anti-cancer treatments, such as ionizing radiation (IR) and many chemotherapeutics. As such, there has been intense interest in elucidating the key proteins involved in DSB repair, and the mechanisms by which they function. Such insights would be important for a better understanding of the DNA damage response in mammalian cells and in developing novel cancer therapeutics. There are two key DSB repair pathways in mammalian cells, non- homologous end joining (NHEJ) and homologous recombination (HR). There have been numerous insights into the key players in these pathways, and even recently many new proteins were found to play important roles in the recognition, triage and repair of DSBs. DSB repair assays are critical for these research efforts, and better assays are needed which report on repair events that occur at endogenous sites in the genome. I started my laboratory in August of 2012, and during our first 2 years we developed a novel approach to assess DSB repair at genomic loci using next-generation sequencing and automated sequence alignment. In preliminary studies, we created a next-generation sequencing protocol and we demonstrated initial feasibility with an engineered nuclease site in cells. In Aim 1, we will adapt our platform to measure NHEJ at multiple endogenous genomic sites. In Aim 2, we will further engineer our system to measure both NHEJ and HR at a naturally occurring region of flanking sequence homology in the genome. We are proposing to develop a completely novel method to measure DSB repair, which does not require the integration of sequences into the genome, and which does not rely on a fluorescent protein as a reporter. The lack of dependence on a fluorescent protein as a read-out means that DSB repair activity can be assessed nearly instantaneously, and at multiple time-points with fine resolution. The experiments proposed in this R03 will serve as a critical proof-of-principle that both mutagenic NHEJ and HR can be measured at endogenous loci using a next-generation sequencing-based approach. Thus, they are consistent with the R03 grant mechanism which supports the development of new research technology and methodology.